In the related art, in a case in which a weak-phase object such as a biological sample is imaged using a transmission electron microscope (TEM), the contrast of an acquired image becomes weak due to a low spatial frequency component of the observed object. It is important to acquire high contrast from a low spatial frequency component for quickly identifying the range of protein coating in a biologically important organism or structure (an intra-cell composition, a virus, or the like).
In the related art, in the field of transmission electron microscopes, in order to enhance contrast, phase plates inserted into passages of electron beams have been developed. Typically, a phase plate is disposed in a back focal plane of a transmission electron microscope or at a conjugate position of the back focal plane and causes a phase difference to occur by changing the phase of an electron wave passing through an area in which the phase plate is present and the phase of an electron wave passing through the other areas. At the present, while there are various types of phase plates, there is a phase plate of a thin-film transmission type as one thereof.
A phase plate of the thin-film transmission type decreases the intensity of the whole image and introduces additional scattering into a passage of an electron beam. For this reason, a phase noise is induced. For a phase plate of such a type, thickness control of high precision (about 1 nm) is required at the time of manufacturing for achieving a desired phase shift. In addition, a phase plate of such a type is sensitive to a thickness change that may easily occur in accordance with carbon contamination or an excitation chemical change according to an electron beam that is in use.
A phase plate of the thin-film transmission type at the initial period includes an opening in the center area of the phase plate. However, the phase plate having such a structure has a defect of having a phase shift easily changing in accordance with contamination or defects of a surface of the thin film generated in manufacturing. In addition, the magnitude of the phase shift depends on the intensity and the time of incident electrons, and thus, the practical use of the phase plate becomes complicated. Some of such phenomena can be reduced by covering a support frame of the phase plate and the surface of the phase plate with a carbon coating film and further heating the phase plate. However, even by employing such a countermeasure, the temporal stability of the phase shift cannot be improved.
Thus, recently, as illustrated in FIG. 1, phase plates having no opening in the center portions thereof have been proposed (NPTLs 1 to 3). These phase plates have a principle that a non-scattering electron beam formed on a back focal plane of a transmission electron microscope causes a phase shift on a thin film of the phase plate. In an insulating surface layer covering upper and lower faces of the conductive thin film of the phase plate, a portion irradiated with a non-scattering electron beam is in a state in which electrons are insufficient due to secondary electron discharge. For this reason, in the insulating surface layer, a positively-charged area accompanying a negatively-charged area right below thereof is generated. Net charge of this center area generates local net electrostatic potential different from that of a peripheral area for electrons passing through such an area. As a result, phase differences are generated to be different for electrons passing through the center portion and the peripheral portion.